Mutations can arise either by chemical events that directly cause changes in the base sequence of DNA, or they can arise from error-prone DNA synthesis during replication, repair or recombination of DNA. The aim of the proposed project is to develop biochemical and genetic methods for detection of errors made during synthesis of DNA in vitro, and to characterize biochemically the error-prone step which has been proposed to account for mutagenesis in vivo. For detection of error-prone DNA synthesis, the well-characterized in vitro conversion of circular, single-stranded DNA of bacteriophage G4 to duplex form will be utilized. Incorporation of incorrect nucleotides during this DNA synthesis will be detected in two ways: (1) Endonuclease assay: Incorporation of noncomplementary nucleotides during synthesis on the G4 template will create mismatches in the duplex product of the reaction, making it susceptible to cleavage by purified endonucleases that recognize mismatches. (2) Genetic assay: Error-prone synthesis on a template strand carrying a base substitution (amber) mutation should yield some newly-synthesized strands containing the wild-type nucleotide sequence, detectable by a transfection assay. This in vitro system for detection of error-prone DNA synthesis will be used to test the hypothesis that mutagenesis induced in certain organisms by various agents is due to an inducible, error-prone repair process. In addition, this system will be used to purify and characterize proteins that influence the fidelity of DNA synthesis, and to investigate whether malignancy, or genetic predisposition thereto, is associated with error-prone DNA synthesis.